Autotransporters are a large family of outer membrane proteins that play key roles in the
pathogenesis of many infectious diseases. Their virulence function is dependent on their
correct assembly at the bacterial cell surface. This requires a C-terminal b-barrel domain to
be folded and inserted into the outer membrane to facilitate translocation of the N-terminal
passenger domain to the bacterial cell surface where it exerts its virulence function. The
autotransporter of interest, antigen 43 (Ag43) is found in uropathogenic Escherichia coli and
mediates biofilm formation by cell-cell autoaggregation after being processed from its
cognate b-barrel only to remain attached to this domain in a non-covalent manner. To date,
the majority of Ag43 research has focused on the passenger domain, leaving the structure and
function of the Ag43 b-barrel domain uncharacterized. Using biochemical and biophysical
methods, the current study showed the fifth extracellular loop of the Ag43 b-barrel domain,
which connects b-strands 9 and 10, is required for the correct folding and function of the
Ag43 passenger domain. We also developed an in vitro refolding assay for Ag43. The results
are the first to ever provide evidence that processing of the Ag43 passenger domain occurs
via an autocatalytic cleavage mechanism. These findings provide valuable insights into the
assembly pathway of Ag43 and reveal new potential therapeutic targets in treating UPEC
infections.